FI102724B - Process for the preparation of a complex consisting of a biologically active substance and a lipid - Google Patents

Abstract

Methods and compositions are described for nonliposomal lipid complexes in association with toxic hydrophobic drugs such as the polyene antibiotic amphotericin B. Lipid compositions are preferably a combination of the phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in about a 7:3 mole ratio. The lipid complexes contain a bioactive agent, and may be made by a number of procedures, at high drug:lipid ratios. These compositions of high drug:lipid complexes (HDLCs) may be administered to mammals such as humans for the treatment of infections, with substantially equivalent or greater efficacy and reduced drug toxicities as compared to the drugs in their free form. Also disclosed is a novel liposome-loading procedure, which may also be used in the formation of the HDLCs.

Description

102724

This application is a partial extension of U.S. Patent Application No. 069,908, filed June 6, 5, 1987, which in turn is a partial extension of U.S. Patent Application No. 022,157, filed March 5, 1987.

This invention relates to methods of making drug-related lipid complexes having high drug content in relation to lipid (high drug, lipid complexes, or HDLC). Such complexes are generally essentially equivalent or more potent than the same drug in free form, yet have lower toxicity.

Complexes (HDLC) with high drug content relative to lipid can be prepared by essentially similar techniques to liposomes.

Yet another embodiment of the invention, a novel process for the formation of HDLCs without the use of organic solvents, is disclosed. The drug attachment or coupling to the HDLCs is performed via ethanol or an aqueous intermediate.

The essential features of the invention are set forth in the appended claims.

Several drugs useful in the treatment of the disease show toxicity in the patient; such toxicities may be heart attacks such as the anti-tumor drug doxorubicin, or renal toxicity such as aminoglycosides or polyethylene -. (Taylor et al., Am. Rev. Respir. Dis. 1982, 125: 610-611). Because * · amphotericin B is a hydrophobic drug, it is water insoluble and is obtained commercially as a colloidal dispersion in deoxycholate, a detergent used to suspend it, and which itself is toxic. Amphotericin B methyl ester and • · · *. amphotericin B has also been shown to be active against HTLV-II / LAV virus, a retrovirus in the lipid envelope, and demonstrated in immune deficiency (AIDS): *** (Schaffner et al., Biochem Pharmacol. 35: 4110-4113 (1986). In this study * ·. amphotericin B methyl ester ascorbic acid salt (water soluble) and amphotericin B were added to separate HTLV-III / LAV infected cell cultures and viral replication was determined. The results showed that methyl ester of amphotericin B and amphotericin B protected target cells against the cytopathic effects of the virus 2,102,724, as has been shown for herpes virus (Stevens et al., Arch. Virol., 1975, 48: 391).

In previous studies, lipid-containing liposomes were used to reduce the toxicity of the enclosed drug by seeking to increase the lipid concentration in the formulations to buffer the drug's toxicity. Applicants have surprisingly found that low lipid material is actually the most effective in reducing toxicity. When creating the HDLC of the invention by MLV, the result may be a mixed population of HDLC and MLV; these formulations are those employing about 5 to 25 mol% of drug (amphotericin B), with an increase in the proportion of HDLC when the molar 10% of drug is increased. Preparations using 25 mol% to 50 mol% of drug are essentially HDLC free of liposomes. Alternatively, formulations containing 5 mol% hydrophobic drug or less are predominantly liposomes with a slight amount of HDLC. Separation of HDLC from a heterogeneous population, if necessary, can be accomplished using any known separation technique, eg density gradient centrifugation.

The methods used to generate these HDLCs may be essentially the same as those used to form the liposomes, but using high drug: lipid ratios in this invention produces more HDLCs than liposomes having an unexpectedly high toxicity reduction compared to 20 na for liposomal stimulation.

This invention provides HDLC (complexes with high drug content relative to lipid) systems consisting of lipids and bioactive agents

. ···. including medicines. Such HDLCs may contain phospholipids such as DMPC

And DMPG, preferably in a 7: 3 molar ratio, or saturated phospholipids or fatty acid phospholipids. Preferably, the bioactive agent is a drug, such as an antifungal drug, such as ny Starin or amphotericin B. Preferably, the mole percent of the drug present is from about 6 to about 50 mol%, more preferably from about 30 to about 50 mol%. The pharmaceutical formulations of HDLC • · · *. · ", Preferably including a medicament such as amphotericin B, are made ... with pharmaceutically acceptable carriers or diluents and these ····. ···. Such compounds are used in the treatment of infectious diseases such as home infections by administering them to mammals such as humans.The HDLC-containing compounds of this invention include those which are essentially free of liposomes that encapsulate the drug. 10% by weight, preferably not more than about 5%, and most preferably not more than about 3%.

3, 102724

The present invention discloses several methods for preparing HDLC; e.g. techniques for first dissolving a drug, especially amphotericin B in a solvent such as DMSO or methanol. The lipid (preferably DMPC: DMPG in a 7: 3 molar ratio) is dissolved in a solvent such as methylene chloride and the lipid and drug solutions are mixed. The solvents can be evaporated under reduced pressure to give a thin lipid drug film. The membrane may be hydrated in an aqueous solution such as saline, PBS or glycine buffer to form HDLC. Alternatively, the aqueous solution may be added to the solvent-containing drug and lipid phase prior to evaporation of the solvent. Alternatively, the resulting dry lipid drug film may be resuspended in a solvent such as methylene chloride and evaporated again under reduced pressure prior to hydrogenation of the film. The dehydrogenation process may also be used; in this method, the dry lipid drug film is dehydrated to form a flake which is hydrogenated with an aqueous solution.

In an alternative method of generating the HDLC of the invention, lipid particle-crystals (or liposomes) containing a bioactive agent (drug, e.g., antifungal polyene, such as amphotericin B) made by the MLV process and containing from about 6% to about 50 moles % amphotericin B is formed and then the particles (or liposomes) are subjected to a heating cycle at about 25-60 ° C, preferably about 60 ° C. In such a cycle, highly organized and less toxic amphotericin 20B / lipid complex is formed.

In the context of the invention, the drug-lipid absorption spectra technique can be used. . ·. to determine the toxicity of the complex (e.g., antifungal polyene such as amphotericin B). The absorbance spectrum of a drug is specific for each drug; medicine. the mark may be a peak or a series of peaks in ultraviolet or visible region.

The characteristic peak of amphotericin B (shown in Fig. 8, dissolved in deoxycholate • · · · *) between about 300 and 500 nm and has characteristic peaks, most typical of these being · · * * · * · * the peak is that which appears in No. 413. The attenuation of this peak when the drug complexes with the lipid can be used quantitatively as a measure of * · HDLC toxicity. Ts. the degree of toxicity can be measured as the intensity of the absorbance peak: *: *: 30.

'*: Figure 1 is a graph depicting acute toxicity as measured by LD50: molar percentage of amphotericin B in the preparation.

Figure 2 is a differential scanning calorimetry spectrum of an HDLC preparation (MLV method) containing 25 mole% amphotericin B.

4, 102724

Figure 3 is a graph of an isopyclic gradient of HDLC (MLV method) made with 50 mol% of amphotericin B. Lipid (solid line); amphotericin B (dashed line).

Figure 4 shows graphical representations of the X-5 protein diffraction of liposomes and HDLC (MLV method) containing 5 and 50 mol% (respectively) of amphotericin B.

Figure 5 shows a 31 P NMR spectrum for HDLC preparations (MLV method) containing 25 and 50 mol% of amphotericin B.

Figure 6 is the absorbance spectrum of free amphotericin B dissolved in deoxycholate.

Figure 7 is the absorbance spectrum of 5 mole% amphotericin B (a), 25 mole% amphotericin B (b), 25 mole% amphotericin B (c) and 50 mole% amphotericin B (d), 7: 3 DMPC: DMPG.

Figure 8 is the absorbance spectrum of 25 mole% amphotericin B 7: 3 DMPC: DMPG both before (a) and after (b) heating for 10 min at 60 ° C.

Figure 9 is a DSC spectrum of 7: 3 DMPC / DMPG with 25 mole% amphotericin B both before (a) and after (b) heating cycle compared to pure DMPC / DMPG (c) .

Figure 10 is a 31 P NMR spectrum of DMPC / DMPG complexes at 7: 3 molar ratios containing 25 mol% of amphotericin B before (a) and after (b) heating cycle: 60 ° C.

• · «

Non-liposomal high drug-lipid complexes (HDLC) having unique properties; and methods for their preparation are described. These HDLCs contain a bioactive agent, such as a hydrophobic drug, whose efficacy when administered to the body of the HDLC may be substantially equivalent to or greater than that of the drug administered either in free or liposomal form. : T: Such HDLCs have a high drug: lipid to molar ratio (greater than about 5 mol% drug). The present invention surprisingly demonstrates that complexes having. less lipid, as in HDLC systems, has reduced toxicity compared to liposomal systems.

HDLC refers to a drug-related lipid in particulate form, such particles being non-liposomal in nature. Using the MLV method 102724, such HDLCs are characterized by, for example: (1) freeze-splitting electron micrographs (Deamer et al., Biochim. Biophys. Acta, 1970, 219: 47-60). show non-liposomal complexes; (2) internal volume measurements (Deamer et al., Chem. Phys. Lipidis, 1986, 40: 167-188), which show a substantially internal zero volume and are therefore non-liposomal; (3) differential scanning calorimetry (DSC) (Chapman, D., in Liposome Technology, Gregoriadis, G., ed., 1984, CRC Press, Boca Raton), which does not indicate a lipid bilayer precursor or main transition step ; (4) 31 P NMR (Cullis et al., 1982, Membrane Fluidity in Biology, Academic Press, Inc., London & N.Y.), which refers to the characteristics of highly immobilized lipid (broad-fold); and (5) X-ray diffraction data (Shipley et al., Biomembranes, 1973, Chapman, D. and Wallach, D., eds., Vol. 2: 1, Academic Press, Inc., London & NY :), which express a gel phase lipid. . It is also characteristic of these ice systems that the drug is completely bound to lipid, which has been demonstrated by density gradient centrifugation. With this technique, the gradient is centrifuged at an elevated force (about 230,000 x g) for about 24 h. This prepares all components in the gradient to reach their density equilibrium position. The elution profiles of these systems show overlap between drug and lipid peaks, indicating that all drug is lipid-associated.

One pine of the present invention is a drug: lipid ratio, which forms HDLC, which results in substantially the same or greater potency of the drug, generally reducing acute toxicity as measured by LD50 in mice (Figure 1). Applicants have found that about 6-50 mol% of a hydrophobic drug achieves the following requirements, with a preferred ratio of about 15-50, more preferably about 25-50, most preferably about 25-35 mol% of the hydrophobic drug. When the concentration of drug: 25 exceeds about 6 mole% and reaches 25 mole%, mixed populations of li: Y: posomes and HDLC are formed. Within this range, as the drug mole percentage approaches 25, a higher percentage of the structures are HDLC rather than Liposome. At a drug concentration of about 5 mol% and less and at lower lipid levels. known in the art as "critical micelle concentration" present in HDLC / - · · · 30 liposome mixed populations, but mainly liposomal structures.

• · · • · · ·. * ··. Characterized by the above-mentioned HDLCs are the various drug-lipid dispersions observed by density centrifugation. The separation of drug-lipid (DMPC: DMPG 7: 3 molar · * *) complexes on isopycnic sucrose density columns shows the formation of banding, which depends on the drug: lipid ratio and the method of preparation. In systems containing 5 mol% drug by MLV, two major zones of the material are detected; one with liposomes and one with 6,102,724 drug bound to lipid (HDLC). Preparations containing 25 and 50 mole% of drug showed a single band, with most drug bound to lipid. Surprisingly, these low lipid mole percent / higher drug mole percent systems were less toxic. Figure 1 shows LD50 (mg / kg) in mice as a function of 5% drug amphotericin B in the preparation. The LD50 increases in the range of 5-50 mole percent drug, then drops at 60 mole percent. Also illustrated is LDSO for a commercially available form of this drug; Fungizone, about 3.5 mg / kg. In vitro lysis of red blood cells (haemolysis) shows the same toxicity.

Internal volume studies (surrounded solution (in pm) per pmol of lipid) performed on MLV preparations of drug-lipid interfaces at varying drug mole percentages show the unusual nature of 25 mole percent and higher (HDLC) formulations; these systems do not contain the solute and therefore are not liposomal. Freezing electron micrograms of these systems show liposomes at 0 and 5 mol% of drug, but non-liposomal HDLCs at 25 and 50 mol% of drug.

Differential scanning calorimetry curves performed on MLV formulations by varying the mole percent drug exhibit the same phenomenon; i. that the spectrum shows transition peaks characteristic of an undisturbed bilayer 20 lipid state at 5 mol% drug but no transition peak at 25 mol% drug at 25 mol% drug (Figure 2). In the case of 25% medicine '. all lipid is completely incorporated into the drug, i.e.. lipid acyl chains are not. : free for cooperative migration. This information confirms density centrifugation data showing the double peak for lipid-bound drug and free li-25 at 5 mol% drug concentration (Figure 4) but a single peak, *; when lipid is drug bound at 25-50 mol% drug concentration * * · * (Figure 3).

X-ray data at 5 mol% (MLV method) indicates a low temperature gel phase lipid that migrates to the liquid crystal phase at a temperature of 23 ° C in nature. However, at 50 mole percent of the drug, the lipid is in the gel phase at all temperatures; there is no transition due to the lipid being tightly bound to the drug (Figure 4). Similar data from P-NMR studies indicate the absence of free lipid signal from these higher (25 and 50) mole:% drug formulations (HDLC); the low field shoulder / high field 7 102724 peak, characteristic of this type of lipid, is absent (Figure 5), while it is visible in O and 5 mol% drug samples.

Lipids that can be used to make lipids include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidyl-5-glycerol (PG), phosphatidic acid (PA), phosphatidylinositol ( (SMP) and the like, alone or in combination. Saturated phospholipids such as hydrogenated soy PC may also be used. Phospholipids may be synthetic or derived from natural sources such as egg or soy. Yet another lipid compound is saturated fatty acids such as myristic acid. In preferred embodiments, the phospholipids employ dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidyl glycerol (DMPG) in any molar ratio, from about 99: 1 to about 1:99 DMPC: DMPG, preferably about 7: 3 molar ratio. DMPC and dimyristoyl phosphatidylserine (DMPS) may also be used in combination. However, DMPC can be used alone. Lipid complexes may also have a steroid-15 component as part of the lipid phase, such steroids may be cholestrol, polyethylene glycol derivatives of cholestrol (PEG-cholesterols), coprostanol, cholestanol, cholestane, organic acid derivatives of sterols, such as cholesterol succinate. Organic acid derivatives of tocopherols can also be used as complexing or liposome forming agents, such as alpha-toco-20 ferric hemisuccinate (THS). Both CHS- and THS-containing complexes and their tris salt forms can generally be prepared by any known method for preparing liposomes containing these sterols. See in particular: The method of Janoff et al., U.S. Patent No. 4,721,614, issued Jan. 26, 1988, which: is entitled "Steroidal Liposomes" and Janoff et al., PCT Publication No. 87/02219, Jul-1; **: 25 lanes, April 23, 1987, titled "Alpha-Tocopherol Based Vesicles", filed ♦♦ · September 24, 1986, the related parts of which are incorporated herein by reference.

Hydrophobic bioactive agents such as pharmaceuticals may be used in the present invention. HDLCs are formed with hydrophobic bioactive agents such as, but not limited to, polyene antibiotics, e.g., amphotericin B and nystatin.

Organic solvents, such as in the general preparation of liposomes, may also be used in the preparation of HDLC. Suitable organic solvents include those having inter-I · · 'medial polarities and dielectric properties (having polar intermediate "" to opposite electrical charges which dissolve lipids and include, but are not limited to, chloroform, methanol). , dimethylsulfoxide (DMSO), methylene chloride, and: solvent mixtures such as chloroform: methanol (70:30) and benzene: methanol (70:30).

s 102724

As a result, solutions are formed which are described as mixtures in which the components are uniformly distributed throughout and contain lipids. Solvents are preferably selected for their bio-miscibility due to their low toxicity and non-flammability. When dissolving a drug, especially amphotericin B, DMSO 5 is preferred as it is best soluble in DMSO. DMSO can be replaced with methanol by simultaneously increasing the solvent volume. Methylene chloride is preferred for lipid dissolution because of its low toxicity to humans.

Aqueous solutions such as distilled water (e.g., USP water for injection), saline or aqueous buffers can be used in the hydrogenation step of HDLC formation. Aqueous buffers which may be used, but are not limited to, include buffered saline solutions such as phosphate buffered saline ("PBS"), tris (hydroxymethyl) -aminomethane chloride ("tris") buffers or glycine buffers at pH about 7.0. - 7.5, preferably 7.2. Sonication can be performed during the HDLC formation step. Such a procedure is performed in a bath sonicator for about 15-30 minutes at 25 ° C, about 50-60 Hz.

The formed HDLCs can be sorted by size by dropping through a filter according to the method of Cullis et al., PCT publication no. No. 88/00238, issued Jan. 16, 1986, the disclosures of which are incorporated herein by reference. Such sorting by size 20 allows for a homogeneous population of cuttings. size. For example, HDLC filtering can be accomplished through a complex path or directly through a membrane filter, such as a polycarbonate filter.

· ;;; One method of forming HDLC is the MLV method, wherein the bioactive • ♦ Y Y * substance (e.g., drug) is dissolved in methanol in a flask to which is added a lipid such as *. *. DMPG in chloroform. After evaporating the solution in a rotary evaporator under reduced pressure, the dried membrane is resuspended in PBS and sonicated in a bath sonicator (about 25 ° C for about 30 min, about 50-60 Hz) to clear. At a drug ratio of about 6 and higher (about 60 mole percent), the formulations are non-liposomal HDLC-r-30 structures that are essentially free of liposomes. Toxicity of HDLC preparations; · * Dependent on drug-to-lipid ratio, preparations with higher drug-to-lipid ratios::: (about 16 to 50 mol% drug) show less acute toxicity:; than preparations with a lower drug-to-drug ratio (about 6 to 15 mol% drug). As discussed above, formulations containing about 5 mole percent of 35 drugs are mainly liposomal.

9 102724

Another method is a modified MLV method wherein the drug-lipid film produced above is dried in a clock vessel under reduced pressure to produce a drug-lipid flake. This flake is ground to a powder which is homogeneously hydrogenated when an aqueous solution is added. This process involves dissolving the drug in an or-5 organic solvent such as DMSO or methanol, followed by mixing with lipid (most preferably 7: 3 molar ratio DMPC: DMPG) in methylene chloride. The mixture is then dried under reduced pressure to a film which is then dried, for example in a clock vessel under reduced pressure. The resulting dried powder is hydrogenated with aqueous saline and heated to produce a drug-lipid suspension. As discussed above, the formation and concomitant degree of toxicity of HDLC, liposomes and mixtures thereof are dependent on the drug-lipid ratio.

Other methods may be used to form the HDLC. One such method is the SPLV method wherein the drug is dissolved in a solvent such as DMSO. The lipid (such as 7: 3 molar DMOC: DMPG) in a solvent (e.g., chloroform or methylene chloride) is dried to a thin film under reduced pressure and an organic solvent such as methylene chloride is added to the lipid. An aliquot of the drug (e.g., amphotericin B) in solution is added to the lipid suspension and the resulting mixture is sonicated to clear. A buffered aqueous solution (e.g., buffered saline) is added and the mixture is again placed under reduced pressure to remove methylene chloride. The resulting paste is further hydrolyzed with PBS and sonicated to clear. As in the above method, the resulting preparation is HDLC or liposomal, depending on the drug-lipid molar ratio used. When used in mice, the LD no showed lower toxicity at higher drug: lipid ratios.

Yet another method is the modified SPLV method, wherein the drug is mixed with an organic solvent (e.g., DMSO) and a lipid (e.g., DMPC: DMPG), which is in solution, and a buffered aqueous solution (e.g., PBS) is added. ) followed by evaporation of the solvent under nitrogen while sonication. More PBS is added and the resulting · · · '· [· ’solution is filtered, preferably through a 5 micron filter, then centrifuged in V; about 10,000 x g for about 10 min. The supernatant solution is removed, discarded and the pellet suspended in aqueous solvent (PBS). Wash a second time for centrifugation ····. ···. club, the resulting pellet is resuspended in PBS. As in the formulations above, the acute toxicities of the formulations are directly proportional to the drug: lipid * ratio; i. the higher the ratio (about 60 mole% of drug), the less * the preparation is toxic.

10 102724

Another method involves dissolving the drug in acidified anhydrous ethanol by sonication. The lipid (e.g., 7: 3 molar ratio DMPC: DMPG) dissolved in a solvent (e.g., benzene: methanol) is lyophilized, then mixed with Vortex in an aqueous solvent (e.g., PBS) and acidified alcoholic alcohol solution is added and mixed at room temperature. The preparation is then lyophilized and re-hydrogenated with aqueous buffer. Alternatively, the solvent may be evaporated, whereupon the ice lipid-drug membrane moiety may be hydrogenated with an aqueous solution. Again, lower acute toxicities are associated with formulations with higher drug to lipid ratios. At drug concentrations of about 5 molar percent or less, the method produces mostly liposomes when compared to use of about 6 molar percent or higher, producing mainly HDLC. When centrifuging the preparation in a density gradient, it is prepared that all lipid is drug bound. The preparation can then be lyophilized to remove the ethanol and re-hydrogenate in an aqueous solution such as distilled water.

Yet another method of forming the HDLC of the invention is to use a homogenization step rather than sonication. For example, HDLCs can be made according to the SPLV method, where a drug (e.g., amphotericin B) can be added to a suitable solvent (such as DMSO) and mixed with a mechanical stirrer to dissolve all drug. If necessary, the solution can then be filtered to remove any insoluble drug particles. At a 7: 3 molar ratio, DMPC: DMPG can then be dissolved in a suitable solvent (such as methylene chloride) and then lyophilized into the drug DMSO solution. An aqueous solution, such as brine, is then added to the mixture, and the organic solvents are removed by rotary evaporation at about ·: 35-45 ° C. After removal of the solvent, the resulting drug-lipid mixture is diluted with an aqueous solution such as saline blade The HDLC suspension is ground using a homogenizer Any homogenizer or colloid mill that grinds particles must be acceptable for this process, but preferably a Gifford Wood colloid mill is used until milling is achieved until an acceptable · · · 11 ", for example 90% of the particles are less than 10 µm in diameter, preferably in the size range of about 4 to 10 microns, or about 15 to 30 minutes. The particles can be recycled through the mill one or more times depending on the desired size: and HDLC particles can be analyzed for size distribution. <! · „using Malvem particle If necessary, larger and smaller particles can be removed by any known particle separation method, such as filtration. Such a procedure preferably yields particles having a size of 0.2 to 10.0 µm in diameter.

π 102724

Other methods known to those skilled in the art for the formation of liposomes may be used to practice the present invention; The HDLC invention is not limited to the above methods for their formation.

HDLC preparations and liposomes derived from the novel process of this invention may be used therapeutically in animals (including man) for the treatment of a variety of infections or conditions requiring: (1) repeated drug administrations; (2) continuous administration of the drug in its bioactive form; or (3) reduced toxicity, and at the same time essentially similar or greater potency of the free drug in question. Such conditions include, but are not limited to, fungal infections, both local and systemic, such as those that can be treated with antifungal agents such as nystatin and amphotericin B, and viral diseases, immune deficiency (AIDS), and herpes. Further, the formulations of the invention are stable in aqueous solutions.

The compounds of the invention may be used to treat asthma by administering an aerosolized aqueous liposome or HDLC suspension to the lungs. For example, liposomes or HDLC may be suspended in a suitable solvent which may be aerosolized by a pneumatic or ultrasonic nebulizer, or more preferably by a encapsulated nebulizer operating under a gas pressure from a fluorocarbon pellet. Other inhalation systems, such as those in which the liposomes or HDLC are administered in particulate form, either as a dry powder or as a suspension in a suitable carrier system; 20 where, are acceptable. After the aerosol has been formed, it evaporates into the mouth. part of the propellant-containing solvent in expansion evaporation and is replaced. : Humidity in the respiratory tract leading to particulate deposition.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of this invention.

Example 1 * · * .v Amphotericin B (10 mg; total drug 5 mole%) was added to 100 mL of meta · · · v; zero in a 500 ml round bottom flask and sonicated until clear. This sonication step was performed in a bath sonicator for about 15 min. ···. At 25 ° C, about 50-60 Hz. Dimyristoyl phosphatidylcholine (DMPC) (100 '· 30 mg in 1.0 mL of chloroform) was added as well as 42 mg dimyristoyl phosphatidyl glycerol (DMPG) (in 0.42 mL of chloroform). The resulting dispersion was dried by rotary evaporation at 60 ° C under reduced pressure to give a thin film to the flask. The membrane was resuspended in 4.0 ml PBS, transferred to a glass tube and sonicated to clear.

, 2 102724

The above example was repeated with 16.7, 20, 25, 33, 50 and 60 mol% of Amphotericin B. The results of acute toxicity studies (LD50), which measure the dose of drug that causes 50% death in mice, were higher when up to 50 mol% of drug was added to amphotericin B.

Example 2

Amphotericin B (140 mg; total drug 5 mol%) was added to 1.5 mL of dimethyl sulfoxide (DMSO). DMPC (1400 mg) and 600 mg DMPG were dissolved in 50 ml methylene chloride and the two solutions were transferred to a flask. The solution was stirred until clear and then dried on a rotary evaporator under reduced pressure to form a membrane in a flask, then dried for 1-4 days in a clock vessel. After such drying, the film which had formed dry flakes was ground to a powder and mixed with 100 mL or 50 mL of 0.9% saline, or 50 mL with USP water for injection. The resulting suspension was heated at 37 ° C for one hour.

The above example was repeated using methanol rather than DMSO as the suspending solvent for amphotericin B and 10, 16.7 and 33 mol% of amphotericin B. 1g and 2g of egg phosphatidylcholine were used instead of 7: 3 molar ratio of DMPC: DMPG.

Example 3: Amphotericin B (100 mg; total drug 5 mol%) was dissolved in 2.0 ml DMSO. DMPC (100 mg in 1.0 mL) and DMPG (42 mg in 0.42 mL) were dissolved in one bottle of chlorophone and the chloroform was removed by rotary evaporation under reduced pressure. pressure. Methylene chloride (20 ml) was added to the flask, followed by addition of 0.2 ml of a stock solution of amphotericin *** ricin B. This suspension was sonicated under clear (about 1 25 min) conditions as described in Example 1. PBS (0.3 mL) pH 7.2 was added, and methylene chloride was then removed under a stream of nitrogen while sonication. The resulting · · · paste was resuspended in 10.0 mL PBS and centrifuged at 10,000 x g for 10 min followed by bath sonication for approximately 20 minutes.

• · · ·

The above example was repeated using 16.7, 20, 25, 33, 50 and 60 mol% -30 centimeters of amphotericin B.

13 102724

Example 4

Amphotericin B (140 mg; total drug 5 mol%) was dissolved in 1.5 volumes of DMSO. Egg phosphatidylcholine (EPC) (2.0 mg) was dissolved in 50 g of methylene chloride and the two solutions were mixed in a flask. PBS (8.0 mL) was added and the solvent removed under a stream of nitrogen while sonicated according to the method of Example 1. PBS (200 mL) was added.

The above example was repeated using 10 and 20 mole% amphotericin B.

EXAMPLE 5 Amphotericin B (140 mg; total drug 5 mol%) was dissolved in 1.5 incubated DMSC. DMPC (1400 mg) and 600 mg DMPG were dissolved in a 500 µm round bottom flask with 50 g methylene chloride. The two solutions were mixed in a flask and 10 mL USP water was added for injection at 37 ° C. The solvent was evaporated using a stream of nitrogen while sonicated according to the method of Example 1 and then 50 ml of water for injection, USP, was added and the solution was warmed to 37 ° C for 30 min. The above example was repeated using 10 and 16.7 mol% of amphotericin B, followed by filtration of the solution through a 3 micron direct flow polycarbonate filter available from Nucleopore.

Example 6 Amphotericin B (140 mg; 16.7 mol% total drug) was stirred in 50 volumes of methanol and stirred for 15 min to form a solution, then filtered through a 0.22 micron flow-through filter (Miscellaneous Cellulose and Acetate Nitrate • · * ....) Greetings) through Millex. DMPC (350 mg) and 150 mg DMPG were dissolved together with 50 g of methylene chloride and mixed with the filtered solution. The solvents were evaporated in a stream of nitrogen while sonicated according to the procedure of Example 1. PBS (50 mL) was added. Half of the preparation (25 ml) was lyophilized as standard. according to dilyophilization methods.

···· t 11

Example 7 'To 10 ml of anhydrous ethanol was added 10 μΐ of IN hydrochloric acid. Amphotericin B (20 mg; total drug 5 mol%) was added to acidified ... · 30 ethanol and the mixture was sonicated under the conditions of Example 1 except that the sonication time was 30 s to 60 s. DMPC (200 mg) and DMPG (42 mg) ) was placed in a test tube supplemented with 102724 14 benzene: methanol (70:30) solution and lyophilized as in Example 8. The dried lipid was mixed with 2.0 ml PBS and the mixture was vortexed. Amphotericin B solution (0.2 ml) was added to the lipid and the mixture was stirred overnight. The resulting DMPC: DMPG MLVs (200 μΐ) were plated in sucrose density gradient-5 and centrifuged at 230,000 x g for 24 h at 22 ° C. The results are shown in Figure 7; all amphotericin B was associated with the lipid, a broad distribution where the peak of the gradient contained most of the lipid and the major peak of amphotericin B was at the bottom of the gradient. Alternatively, the resulting DMPC: DMPG MLVs were lyophilized and re-hydrogenated with distilled water (2.0 mL).

The above example was repeated using 7, 9, 16.7, 17, 20, 25, 33, 50 and 60 mol% of amphotericin B. At drug mole percentages of 16.7 or more, the majority was HDLC. Density centrifugation gradients for such high drug lipid formulations are similar to those shown in Figure 3, where all drug and lipid are combined to form a single peak.

Example 8 Samples of 25 mole% amphotericin B-DMPC: DMPG were prepared for X-ray diffraction, DSC, freeze-division electron microscopy and 3IP-NMR studies as follows: 7: 3 molar ratio of DMPC: DMPG (44 mg total female lipid) ) and 20 mg of amphotericin B (25 mol% of amphotericin B) sus-20 was suspended in chloroform: methanol (70:30 v / v) and evaporated under reduced pressure at 55 ° C to a thin film on the sides of the flask. The membrane was hydrogenated with 8.0 mL of 20 mM He nests, 250 mM NaCl, pH 7.2 at 22 ° C by vortexing with glass beads. The preparation was centrifuged at 10,000 x g for 10 min, the supernatants decanted and the pellet resuspended in 1.0 ml Hepes / NaCl buffer. The preparation was then sonicated in a bath sonicator for 20 min at 25 ° C, 50-60 Hz.

• · · • · · i i i

The above procedure was repeated using 0, 5 and 50 mole% amphotericin • · · B.

v: Example 9

To determine the capturing volume of amphotericin B-containing liposomes and HDLC, the following procedure was used: DMPC (100 mg) and 42 mg DMPG, each in chloroform, were combined with 10 mg of amphotericin B (25 molar). %) contained in methanol (0.1 mg / ml amphotericin: B) in a flask. The solvent was removed under reduced pressure at 37 ° C. 15 102724 PBS (3.7 mL) were added with 0.3 mL of dilute 3H-inulin solution in distilled water, shaking on dry lipid film. An even portion (10.2 ml) of this solution was placed in a scintillation mixture and counted for radioactivity in a beta scintillation counter. From the second part, phosphate was determined according to the Bartlett method, J. Biol. Chein., 5, 1959, 234: 466-468. The lipid drug suspension was centrifuged at 10,000 x g for 10 min, the supernatant discarded, and the pellet resuspended in PBS. The centrifugation and pellet resuspension steps were performed two more times; the last resuspended was collected (100 μΐ) and counted in a beta scintillation counter. From the second part of the final pellet resuspension, phosphate was assayed as above. The percentage of captured inulin was calculated by dividing the final radioactive readings by the initial readings and multiplying by 100. The captured volume (μΐ of captured inulin / μιηοΐ lipid) was also calculated.

The above procedure was also repeated using 0, 5 and 50 mol% of amphotericin B.

15 Example 10

Amphotericin plugs were prepared by drying 15.5 mg DMPC and 6.5 mg DMPG (7: 3 molar ratio) in about 10 ml chloroform at the edges of a 100 ml round bottom flask. Amphotericin B (10 mg) was suspended in 100 volumes of methanol and 100.0 ml of methanolic solution was added to the flask and the lipid membrane was suspended to give 20 molar% of amphotericin B. The mixture was then dried in a rotary evaporator at 37 ° C to form a thin film on the edges of the flask. The film was then hydrogenated with 4.0 mL of 10 mM Hepes, 150 mM NaCl (pH 7.2) using glass beads and sonicated for 30 min to give a final suspension. A sample of this suspension was then heated to 60 ° C for 10 minutes * by immersion in a water bath The resulting suspension was then characterized by DSC,? · 25 NMR and freeze-division electron microscopy.

The above procedure was repeated without heating the sample. This sample was maintained at 22 ° C and also characterized by the above techniques.

• · ·

Example 11 ···· * ··· The materials and methods of Example 10 were repeated using 50 mole% amphotericin B. These systems were characterized by DSC, ESR, and freeze-cleaving: ": electron microscopy.

Example 12 ie 102724 DSC measurements were performed on a Micro Cal MC-1 Unit, which was Micro Callta, Inc., Amherst, MA. 0.70 ml sample volumes of 5-9 mg suspension were injected into the sample cells and the same volume of buffer was used in the reference cells. Samples of moon-5 were lost at either about 26 ° C / hr or 37 ° C / hr. Duplicate determinations of the same sample with the same treatment gave start and stop temperatures that were reproducible at 0.2 ° C. Generally, samples containing amphotericin B were heated to 60 ° C (not hotter) and then cooled to 7-4 ° C for at least 2 h to ensure a consistent sample history.

Example 13 NMR spectra were obtained at 145.7 MHz on a Bruker AM360 wide-diameter NMR spectrometer using 8K data points for printing, a 50,000 Hz scan width and a 20 μβ pulse width corresponding to a 45 ° pulse. Spectra were collected up to 10,000 scans.

Example 14 0.1-0.3 μΐ aliquots of samples were placed between a pair of Balzer (Nashua, NH) copper carrier plates and immersed rapidly at 23 ° C in liquid propane. Samples were sliced and replicated in a dual replication apparatus in a Balzer freezing-splitting unit at 2 x 10 mbar vacuum or higher and at -115 ° C. The replicates were detached in 3N HNCl 2, followed by washing in a tiered series of sodium hypochlorite solutions. These were finally purified in distilled water and collected on copper gratings with a 300> Hex (Polysciences, PA). Replications were observed with a Philips 300 electron microscope, 3000 - 22000 yellow magnifications.

Example 15 • Absorbance spectra (as in Figures 8 and 9) were made by diluting Amphotericin B. . Hepes / NaCl buffer 25 μΜ ^ ΐ liters of amphotericin B. The sample was applied • * ·

Beckman spectrophotometer was sampled and read at 300-500 nm. The sample cuvette • · * * · * 'was read against buffer zero.

Example 16 ···. For the ESR, samples were labeled with a series of substituents of the doxyl steric acids in which the doxyl reporter group was present at various positions along the fatty acid chain Π 102724. Labeling was enhanced by attaching the probe by mixing with an ointment and sonication of an ethanol-containing solution which was dried to a thin film on the edges of the test tube. All samples were labeled to one mole percent.

ESR spectra were recorded on an IBM Instruments ER100D ESR spectrometer with nitrogen gas-5 flow temperature control. An external calibrated term storm probe (Omega Engineering, Inc., Stamford, CA) was used to monitor the sample temperature. ESR spectra were recorded at 10 MW microwave power and 9.11 GHz microwave frequency with a field sweep of 100 G and 100 KHz with a 0.32 G field modulation amplitude. The order parameter was calculated from maximal hyperfine degradation (A max).

Example 17

Amphotericin B (337.5 g) was added to 3375 volumes of DMSO and amphotericin B (100 mg / ml) was stirred to dissolve (about 3 h) using a mechanical stirrer. The mixture was transferred to a stainless steel pressure vessel (5 L volume) and filtered through a 0.22 μηι: η Sartofluor filter and a polypropylene bed filter (Pall Profile, 15 Pall, Inc., Glen Cove, N.Y.).

In a 40 liter pressure vessel, 50.8 kg of methylene chloride was mixed with 225.0 g of DMPC and 99.0 g of DMPG for 3.5 h, so that complete Liu-curing took place. The resulting lipid solution was transferred through a 0.2 M 0.22 micron Sartofluor Teflon filter into a stainless steel treatment tank. The 40 liter pressure vessel was further washed. 55.2 kg of methylene chloride and similarly filtered into a treatment tank. treatment

The lithium tank was set to rotate the lipid solution at 195 ipm and amphotericin B

The DMSO solution was then transferred to a tank. Sodium chloride solution (16.2 L, 0.9% USP) was then added to the tank through a 0.22 μιη Millipak 100 polycarbonate filter.

Using heat exchanger equipment in Saija with a treatment tank, 25 streams of nitrogen soaked with heat were allowed to pass through the tank and the solvent was removed over a period of more than 12 h.

. . The resulting lipid-drug complex was diluted with sodium chloride 0.9% USP (7000 mL), · · M transferred to a Millipak 100 tank through a 0.22 micron filter.

• · · • · ·

The resulting HDLCs were homogenized using a Gifford Wood colloid mill and a rotary block pump. The HDLC was allowed to pass through the head of the colloidal mill with 0.012695 mm 30 aperture settings, 0.7 atm back pressure, 3.0 h to give particles less than 20 microns. The particle size of the HDLC was analyzed with a Malvem particle counter.

102724

1R

Example 18

Lipid (102.6 μιηοΐ total, 70:30 molar ratio DMPC: DMPG) dissolved in chloroform was pipetted into a 500 mL round bottom flask. Nystatin (500mg) was dissolved in 1.0L of inethanol (0.5mg / ml) by sonication on a Branson bath seasonon 5. Dissolved nystatin (5.0 mL) was added to a round bottom flask containing lipid and stirred; the solvent was then removed on a rotary evaporator at 45 ° C for 15 min and the resulting preparations re-hydrogenated in saline (5.0 mL) on a rotary stirrer with glass beads. In light microscope examination, the liposomes were visible.

The above was repeated using 25, 50, 75 and 100 mole% nystatim. Freezing Hal-10 Scanning Electron Microscope Examination A sample of 25 mole% nystatin had no liposomal HDLC.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Add

Claims (10)

A process for the preparation of a complex consisting of a hydrophobically biologically active substance and a lipid, wherein a solution containing the hydrophobic: biologically active substance is admixed with a solution containing the lipid, characterized in that the Sainman mixture is carried out as follows: A.: (a) the lipid is dissolved in an organic solvent of intermediate polarity; (b) the hydrophobic biologically active substance is dissolved in a biologically compatible organic solvent; and (c) the solution from step (a) ) is combined with the solution from step (b) to form a mixture B. (a) The hydrophobic biologically active substance is suspended in an aqueous solution. up; (B) the lipid is suspended in an aqueous solution; . (c) the suspensions from steps (a) and (b) are mixed; and: (d) the product from step (c) is incubated at or above the conversion temperature of the lipid, and that the lipid contains a phospholipid or a fatty acid, that the complex does not contain essential liposomes, and that the concentration of hydrophobically biologically active substance in the complex is at least about 6 mole percent. Process according to claim 1A, characterized in that the biologically compatible organic solvent is an oxygenated solvent, preferably oxygenated ethanol. Process according to claim 1A, characterized in that it further comprises evaporation of the solvent in the mixture from step (c) under reduced pressure and then addition of the aqueous solution in the mixture. 10 Process according to Claim 1A, characterized in that it further comprises adding the aqueous solution to the solution from step (c), evaporating the solvents in the mixture under reduced pressure and then adding the aqueous solution in the mixture. Process according to claim 1A, characterized in that it further comprises drying the mixture from step (c). Process according to claim IA, characterized in that the hydrophobic biologically active substance is an antifungal agent, polyene, preferably amphotericin B or nystatin. Process according to claim IA, characterized in that the concentration of the substance in the complex is about 6 mole percent - about 50 mole percent. Method according to claim IA, characterized in that the lipid is a phospholipid containing dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, preferably in a molar ratio of 7: 3 in the complex. · 9. A process according to claim 1A, characterized in that the hydrophobically biologically active substance is amphotericin B, that the lipid is a phospholipid containing dimyric acid; toylphosphatidylcholine and dimyristoylphosphatidylglycerol, to the concentration of ampho-. r. terisin B in the complex is about 6 - about 50 mole percent and that the mole ratio between. · ·. dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol in the complex are about 7: 3. • ·